Differential WNT4 expression among various subtypes of moyamoya disease results in alterations of microtubule stability

Our study characterized the differential protein metabolism among different subtypes of moyamoya disease (MMD). Moreover, overexpression of WNT4 (wingless-type MMTV integration site family member 4) caused multiple cellular impairments by reducing microtubule stability, which may bring new insight into haemorrhagic MMD.

WNT4, a member of the WNT family located in chromosome 1p36, normally exerts cellular physiological effects through WNT/β-catenin signalling that was observed to be abnormally elevated in thoracic aortic aneurysms, a typical haemorrhagic vascular disease. 3This suggests that overexpression of WNT4 in haemorrhagic MMD may be a new insight into the mechanism.In the validation group, the results of serum ENLISA in the validation group showed that the WNT4 level in HEM (haemorrhagic moyamoya disease) was significantly higher than that in the HC (health controls) and IS (ischemic moyamoya disease), which was consistent with our previous results (Figure 1D, Table S1).We then explored the possible physiological impairment caused by overexpression of WNT4 in haemorrhagic MMD using a variety of in vitro experiments.In immunofluorescence staining, compared with the oe-Vector group, the density of microtubules and cytoskeleton in the oe-WNT4 group was significantly decreased (Figure 2A).Compared with the oe-Vector group, the stability of microtubules in the oe-WNT4 group was significantly reduced by measuring the degree of acetylation (Figure 2C).Considering that this may be the key link between WNT4 and haemorrhagic MMD, we further investigated the WNT4-related signalling pathway to elucidate the relationship between WNT4 overexpression and microtubule abnormalities.The result of the Western blot showed that although the total tau (Tubulin binding protein) expression level was unchanged, p-tau(phosphorylated tau)was increased in the oe-WNT4 group (Figure 2B).This suggests that overexpression of wnt4 may cause an increase in tau phosphorylation.Tau is a microtubule-binding protein that binds to microtubules through its specific domain. 4Previous studies have shown that Wnt/ beta-catenin signaling interacts with presenilin 1 and that levels of tau phosphorylation are significantly higher in cerebrospinal fluid in patients with Alzheimer's disease.{Ref.5 and 6} Although tau is not a structural component of microtubules, hyperphosphorylation can disrupt tua function, leading to microtubule instability and even breakdown.
Microtubules are important components of the cytoskeleton, which function as tracks for intracellular transport. 7Given the critical role of microtubule stability for vascular integrity, we provided in vitro evidence of the cellular physiological impairment caused by microtubule abnormalities in haemorrhagic MMD.The Western blot results showed that the proportion of activated eNOS(endothelial nitric oxide synthase)of the oe-WNT4 group was significantly reduced (Figure S1B).The reduction of activated eNOS will inevitably lead to the reduction of NO, an important cytokine to maintain the normal function of VSNC, which will cause vascular injury. 8In addition, flow cytometry and cell migration assay showed that overexpression of WNT4 also increased cell apoptosis and decreased cell migration (Figure S1C,D).It has previously been shown that a specific increase in ROS in haemorrhagic MMD indicates the abnormal function of cellular mitochondria. 9In contrast, consistent results were observed in our study (Figure S1A).This suggests that microtubule abnormalities caused by overexpression of WNT4 may be involved in the pathogenesis of haemorrhagic MMD through multiple cellular impairment mechanisms.
Immunofluorescence results showed that the density of microtubules and cytoskeleton was restored after WNT4 knockdown by shRNA (Figure 2D).At the same time, the degree of acetylation of microtubules was also significantly increased compared with the shRNA-NC+HEM, suggesting that the stability of microtubules was restored to a certain extent after WNT4 knockdown (Figure 2G).The levels of activated eNOS, apoptosis, cell migration, and ROS were nearly normalized after the WNT4 knockdown (Figure 2D-G).In addition, DDK-1(Dickkopf-1), a selec-tive inhibitor of WNT4, improved microtubule density and alleviated other cell dysfunctions in the oe-WNT4 group (Figure 3; Figure S2).This further confirmed that overexpression of WNT4 could cause microtubule abnormalities and other cellular dysfunctions in haemorrhagic MMD.Hyperphosphorylated TAU loses its ability to stabilize microtubules, which leads to a decline in the stability of cell microtubules and their subsequent disintegration.This results in multiple cellular physiological damage, such as a decrease in activated eNOS, which induces a decrease in NO. β-cat, β-catenin; APC, adenomatous polyposis coli gene; CK1, casein kinase 1; DLV, dishevelled segment polarity protein; eNOS, endothelial nitric oxide synthase; GSK-3β, glycogen synthase kinase 3β; MMD, moyamoya disease; NO, nitric oxide; P, phosphate group; PSEN1, presenilin 1; TCF, T-cell factor; WNT4, wingless-type MMTV integration site family member 4.
To date, the biomarkers and mechanisms of haemorrhagic moyamoya disease are unknown.Our study suggests that overexpression of WNT4 may lead to decreased stability of cellular microtubules, which promotes rupture and haemorrhage of the moyamoya vessel (Figure 4).This study provides a theoretical foundation for the strategy of biomarkers for moyamoya disease typing, which sheds light on mechanistic studies of haemorrhagic MMD.

A U T H O R C O N T R I B U T I O N S
Shihao He, Yuanli Zhao, and Rong Wang conceived and designed the experiments.Shihao He, Zhenyu Zhou, Junze Zhang, Yanru Wang, Ziqi Liu, Xiaokuan Hao, and Xilong Wang performed experiments.Yuanli Zhao, Rong Wang, and Xun Ye contributed reagents, materials, and analytical tools.Shihao He, Zhenyu Zhou, Junze Zhang, Yanru Wang, Yuanli Zhao, and Rong Wang wrote the manuscript.

A C K N O W L E D G E M E N T S
The authors thank the patients with Moyamoya disease and controls who agreed to participate in the study.This study was supported by the Natural Science Foundation of China (82171887 and 82371296 to RW) and National High Level Hospital Clinical Research Funding (2024-PUMCH-E-011 to YL).The above funds provide the testing and processing costs, data collection, analysis, and interpretation of this experiment.

C O N F L I C T O F I N T E R E S T S TAT E M E N T
The authors declare no conflict of interest.

D ATA AVA I L A B I L I T Y S TAT E M E N T
The data presented in the current study are available from the corresponding author upon reasonable request.

F I G U R E 1
Differential analysis and validation of genes for haemorrhagic and ischemic MMD.(A) Principal component analysis (PCA) of the gene data in HEM (N = 20) and IS(N = 20) groups.(Red: HEM group; Blue: IS group).(B) Volcano plot of differentially expressed genes.(Red: upregulation; blue: downregulation).(C) Chordal graph showing the interconnection of the differentially expressed genes.(D) The expression of WNT4 in HC, IS, and HEM serum from different patients.The level of WNT4 was measured by ELISA assay.The abscissa represents the grouping.The ordinate indicates the expression level of WNT4.Results were mean ± SD for HC (N = 20), IS (N = 30), and HEM (N = 30).*p < 0.05, **p < 0.01.

F I G U R E 2
The effects of the expression and knockout of WNT4 on HBVSMC cells.HBVSMC cells were treated with oe-Vector or oe-WNT4.Furthermore, HBVSMC cells were treated with shRNA-NC or shRNA-WNT4, then cultured in SMCM medium with 2% FBS for 24 h, and then cultured in DMEM medium with 2.5% HEM serum for 24 h.(oe-Vector: Control group.oe-WNT4: WNT4 overexpression group.shRNA-NC+HEM: HBCSMC cultured with HEM group serum without knockdown of WNT4.shRNA-WNT4+HEM: HBCSMC cultured with HEM group serum with knockdown of WNT4.).(A) The cell morphology in each group was taken under a light microscope.Bar = 200 µm.HBVSMC cells were treated with oe-Vector or oe-WNT4.The microtubule density in each group was detected by immunofluorescence.Bar = 100 µm.(Green: tubulin; red: actin; blue: cell nucleus; mix: a stack of the three).(B) HBVSMC cells were treated with oe-Vector or oe-WNT4.oe-Vector: Control group.oe-WNT4: WNT4 overexpression group.The expression of WNT4, p-GSK3β, Tau, β-catenin, p-Tau, PSEN1, TUBA, and TUBB from the indicated group was detected by western blot assay.(C) The expression of Acetyl-α-tubulin and α-tubulin from the indicated group was detected by western blot assay.oe-Vector: Control group.oe-WNT4: WNT4 overexpression group.Results were mean ± SD for three individual experiments.The microtubule stability was measured by the acetylation degree of α-tubulin.Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.(D) The microtubule density in each group was detected by immunofluorescence.The ROS levels were detected by DCFH-DA probe fluorescence microscopy.Bar = 100 µm.(E) Flow Cytometric Analysis of cell apoptosis in HBVSMC cells.Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.(F) Cell migration was detected by scratch assay.Bar = 200 µm.Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.(G) The expression of Acetyl-α-tubulin and α-tubulin from the indicated group was detected by western blot assay.The microtubule stability was measured by the acetylation degree of α-tubulin.Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.F I G U R E 3 The effect of WNT4 overexpression on HBVSMC cells after the addition of inhibitor DKK-1.HBVSMC cells were treated with oe-Vector or oe-WNT4, and then one group of cells transfected with oe-WNT4 was cultured in SMCM medium with 100 µg/L DDK-1.(oe-Vector: Control group.oe-WNT4: WNT4 overexpression group.oe-WNT4+DDk-1: WNT4 overexpression group with inhibitor DDK-1).(A) The microtubule density in each group was detected by immunofluorescence.The ROS levels were detected by DCFH-DA probe fluorescence microscopy.Bar = 100 µm.(B, C) The microtubule stability was measured by the methylation degree of α-tubulin.The expression of Acetyl-α-tubulin, α-tubulin, eNOS, and p-eNOS was detected by western blot assay.Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.(D) Flow cytometric analysis of cell apoptosis in HBVSMC cells Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.(E) Cell migration was detected by scratch assay.Bar = 200 µm.Results were mean ± SD for three individual experiments.*p < 0.05, **p < 0.01.F I G U R E 4 Mechanism of microtubule dysfunction caused by overexpression of WNT4 in haemorrhagic moyamoya disease.Overexpression of WNT4 upregulates the WNT/β-catenin signalling pathway, resulting in increased phosphorylation of TAU.